The steroid dehydroepiandrosterone (17 betahydroxyandrost-4-en-3-one, DHEA) and its sulfate derivatives are major steroid adrenal secretory products in humans. DHEA is metabolized to androsterone (3-alpha-hydroxy-5 alpha-androstan-17-one) and estradiol (Estra-1, 3, 5 (10)-triene-3, 17-diol), two major sex hormones in humans. Other metabolites of DHEA include alpha-etiocholanolone (5-betaandrostan-3-alpha-ol-17-one, hereinafter referred to as alpha-ET) and beta-etiocholanolone (5-beta-androstan-3-betaol-17-one, hereinafter referred to as beta-ET) and were, until recently, considered to be inert metabolic end products which were merely conjugated as glucuronides or sulfates and excreted in the urine. The structure of DHEA, alpha-ET, beta-ET and androsterone are shown in FIG. 1. Alpha-ET is the major metabolite of DHEA, and in normal individuals, is excreted in the urine in amounts of about 3-5 mg per day, whereas beta-ET is a minor metabolite in man.
Yen, et al. (Lipids 12:409, 1977) disclosed that DHEA, administered by a variety of routes, decreased the rate of weight gain in a strain of genetically obese mice. Coleman, et al. (Diabetes 31:830, 1982) demonstrated that DHEA treatment had a marked preventive effect on the development of diabetes in either genetically obese or diabetic mice. Furthermore, they indicated that for the maximal beneficial effect, DHEA had to be injested. This suggested that DHEA itself was not the active compound, but that some metabolite of DHEA produced during passage of the steroid through the gastrointestinal tract, was responsible for the activity. Coleman, et al. (Endocrinology 115:239-243, 1984) disclosed that alpha-ET and beta-ET, but not androsterone or epiandrosterone (see FIG. 1 for structure), were four times more effective than DHEA in preventing the development of diabetes in C57BL/KsJ-db/db diabetic mice. Alpha-ET and beta-ET reduced blood sugar, increased plasma insulin concentrations and provided a protective effect on the pancreas, as shown by an increase in the number of granulated islet beta-cells. These results demonstrated that both compounds had physiological significance and were not merely end products of sterol metabolism.
Coleman, et al. (U.S. Pat. No. 4,518,595, incorporated by reference) disclosed that oral administration of DHEA restored hyperglycemia to normal levels and improved glucose tolerance even in severely diabetic mammals. Coleman, et al. (U.S. Pat. No. 4,507,289, incorporated by reference) taught the use of alpha- and/or beta-ET and an estrogen for the treatment of diabetes, obesity syndromes and associated hypercorticoidism. Hypercorticoidism is a condition characterized by excessive functional activity of the adrenal cortex, caused by an excess of adrenalcorticotropic hormone (ACTH). This can lead to Cushing's syndrome, which is characterized by obesity, hypertension, diabetes and other symptoms. It has been proposed that the natural decline in DHEA levels with age produces a relative hypercorticoidism because of the imbalance between circulating levels of DHEA and the corticosteroids. This imbalance may affect a number of regulatory systems. Restoration of DHEA levels may therefore be beneficial in treating some of the diseases of aging, including diabetes and obesity. The activity of alpha-ET and beta-ET suggests that DHEA may actually be exerting its effects through their metabolites. The advantage of alpha-ET and beta-ET over DHEA is that they cannot be converted to estrogenic or androgenic hormones.
Coleman (Endocrinology 117:2279-2283. 1985) disclosed that alpha-ET and beta-ET, when supplied in the diet, have anti-obesity properties. They were effective both in preventing and in arresting the development of obesity as well as in facilitating weight reduction after obesity had been established in diabetic, genetically obese and normal mice. Finally, U.S. patent application Ser. No. 683,423, filed November 21, 1985 of Coleman and Applezweig discloses the use of etiocholanolones for the treatment of obesity, diabetes and other symptoms of hypercorticoidism.
Etiocholanolones have also been shown to participate in the regulation of porphyrin and heme synthesis in hepatic and erythroid cells (Granick, et al., Proc. Nat. Acad. Sci. U.S.A., 57:1463, 1967) and act as inducers of porphyrin synthesis (Wolff, et al., Ann. Int. Med., 67: 1268-1295, 1967).
Gardner and Juneja (Brit. J. Hemat., in press, 1986) treated aplastic anemia patients with either alpha-and/or beta-ET. In this uncontrolled pilot study, 11 of the patients were considered to have acute severe aplasia, and 32 were considered chronic. Seventeen of the patients treated with alpha-ET had hematologic responses lasting longer than six months. (6 out of 11 acute; 11 out of 32 chronic patients). The patients maintained a normal hemoglobin level for long periods of time, and the effects persisted after the alpha-ET was discontinued. However, alpha-ET had been shown to produce acute pyrogenic reactions in man when administered by intramuscular (i.m.) injection (Kappas, et al., J. Clin. Endocr., 16:948, 1956; Kappas, et al., Trans. Assn. Am. Phys., 72:54, 1959). A concomitant dose of prednisolone was therefore used along with therapeutic injections of alpha-ET to prevent the development of fever and alleviate local irritation at the injection sites. Three patients who did not respond to alpha-ET had a hematologic recovery when treated with beta-ET. Moreover, the beta isomer had little pyrogenic effect and a dose of 10 mg of prednisolone was sufficient to counteract any local irritation.
The biological responses obtained in studies with alpha-ET and beta-ET have been correlated with the levels of free alpha-ET circulating in the blood. The use of alpha-ET and beta-ET to achieve therapeutic levels of alpha-ET in the blood has drawbacks, however, because they are rapidly removed from the body by conjugation and excretion, and they are relatively poorly absorbed by the oral route of administration. A means for improving alpha-ET blood levels therefore has the potential to provide superior therapeutic efficiency.
Previous work has suggested that alpha-ET and beta-ET are involved in complex metabolic processes in addition to conjugation and excretion. Administration of alpha-ET and beta-ET i.m. or intravenously [i.v.]to human subjects by Kappas, et al. (J. Clin. Endocr. 16:948, 1956) resulted in the recovery of almost exclusively alpha-ET conjugates in the urine, indicating a transformation of beta-ET to alpha-ET. Bradlow, et al. (J. Clin. Endocr. 27:1203-1207, 1967) found evidence for a rapid oxidation and re-reduction at the C-3 position of a portion of the alpha-ET administered i.v. to human subjects, based on isotopic ratios. A small portion of the alpha-ET also appeared to have a longer lifetime during which it continued to participate in oxidation-reduction systems and, possibly, served as a pool for which other metabolites of alpha-ET were derived. The full nature of the metabolic fate of alpha-ET was unknown at that time and none of the metabolites involved in the process were identified.
The present inventors have now unexpectedly discovered that both alpha-ET and beta-ET administered orally or parenterally (intraperitoneally, [i.p.], i.m. or i.v.) are rapidly oxidized at the C-3 position to form etiocholanedione (5 beta-androstanedione, hereinafter referred to as ET-dione) as shown in FIG. 2, and that ET-dione can serve as a superior source of circulating blood levels of free (nonconjugated) alpha-ET. Once formed, the ET-dione is re-reduced to alpha-ET, which may then be conjugated and excreted. This dynamic interconversion of alpha-ET and ET-dione provides the means of achieving alpha-ET blood levels through the use of ET-dione serving as a pro-drug for alpha-ET. In addition, whereas conjugation and excretion rapidly removes alpha-ET from the blood stream, ET-dione must be reduced to alpha-ET before being eliminated from the body, and may therefore have a longer circulatory half-life than alpha-ET.
Animal and human pharmacokinetic data demonstrate that ET-dione produces increased blood levels of free alpha-ET. This increased bioavailability may be due to improved absorption of ET-dione over alpha-ET. Although the present inventors do not wish to be bound by theory, another possibility is that the superior nature of the ET-dione is due to the fact that the molecule must be reduced to alpha-ET before conjugation-excretion occurs. By administering ET-dione, a reservoir of a precursor to alpha-ET can be established in the blood.
The present inventors have also found that administration of alpha-ET, beta-ET or ET-dione in oil substantially increased the amount of free circulating alpha-ET. This is a further means for increasing the bioavailability of this clinically important steroid.